Organotypic Culture Preparation and AAV Inoculation

Organ of Corti explant cultures were essentially prepared as previously described (Kroll et al., 2019). In brief, cochleae were harvested from p5 Otof-KO mice and organs of Corti of the apico-medial cochlear turn micro-dissected in modified HBSS [HBSS (Thermo Fisher), 10 mM HEPES (Thermo Fisher), 250 ng/ml fungizone (Thermo Fisher), and 10 μg/ml penicillin G (Sigma Aldrich)]. Finally, the explants were mounted on CellTak (BD Bioscience) -coated coverslips in serum-free DMEM/F12 medium [DMEM/F12, 1% N2 supplement (Thermo Fisher) and ampicillin (Sigma Aldrich)] and cultured overnight in a humidified incubator (37°C; 5% CO₂). After 12 h, the coverslips were transferred to 24-well plates for AAV inoculation in growth medium (48 h; DMEM/F12, 1% N2 supplement, 1% ampicillin, 1% newborn calf serum [Thermo Fisher]), with the following AAV titers: AAV2/8: 3.2 × 10¹² genome copies/ml; AAV2/9: 2.4 × 10¹² genome copies/ml; Anc80L65: 6.0 × 10¹² genome copies/ml; PHP.B: 1.3 × 10¹² genome copies/ml; PHP.eB: 5.8 × 10¹² genome copies/ml in 10 μl/well. Subsequently, the growth medium was exchanged 50:50 every 2 days prior to fixation and histological evaluation at DIV9-11.